Marie Curie Scientific Report

1 Enzyme flipping

Finding the target site and associating in a specific orientation are essential tasks for DNA-binding proteins. Proteins can utilize dynamic flipping transitions as they approach the target site to explore multiple orientations. These rapid flipping transitions therefore accelerate the target search process. However, in previous studies it has not been clear how proteins are able to rapidly flip in orientation. Here we probed the mechanism of flipping at the single molecule level, using HIV-1 reverse transcriptase (RT) as a model system. We altered the strength and range of protein-nucleic acid interactions by varying the ionic strength and macromolecular crowding. Our results reveal that while increased ionic strength weakens binding of RT to DNA, increased crowding strengthened the binding affinity. Moreover, we analyzed the flipping kinetics, i.e. the rate and probability of flipping at each ionic and crowding condition. Our data are consistent with a view that DNAbound proteins undergo multiple rapid re-binding events, allowing the macromolecules to reorient themselves in different configurations and engage in different catalytic activities before complete dissociation.

2. Strand displacement synthesis

The process of displacement synthesis, where a DNA polymerase must unwind a nucleic acid duplex without the aid of a helicase, is required for a range of polymerases to properly function in the cell. Viral polymerases, including HIV reverse transcriptase (RT), are known to use displacement synthesis to copy thousands of nucleotides of genetic code. However, the mechanism of displacement synthesis remains unclear. In order to elucidate whether RT actively or passively unwinds downstream duplexes, we measured RT elongation against a series of duplex structures. We find that the both the thermodynamic stability and the backbone content of the duplex influences the rate of DNA replication. We also find that the presence of a nucleic acid flap on the non-template strand increases the efficiency of displacement synthesis. This is the first evidence that RT makes direct contact with the non-template nucleic acid strand. Our results are inconsistent with a passive model of displacement synthesis requiring RT wait for the duplex to melt on its own. Instead, we propose an active mechanism related to some DNA helicases.

3. Dissemination activities

The enzyme flipping research performed under this grant has been submitted to NAR. A second manuscript focused on strand displacement is currently in preparation.

This work has been presented by both the fellow and the PhD student involved in the project (Ganji):

  • At DutchBiophysics 2012, the work was presented as a poster by the fellow.
  • At the NWO CW Study group meeting: Chemistry in Relation to Biology and Medical Sciences, this work was presented as a poster by the fellow
  • At the Institute for Complex Molecular Systems Colloquium, TU Eindhoven, this work was presented in an invited talk by the fellow.
  • At DutchBiophysicsin 2013, 2014, and 2015, this work was presented as a poster and a talk by M. Ganji.
  • At the US Biophysical Society meeting in 2015 and 2016, this work was presented as a poster and a platform talk by M. Ganji.

We are committed to sharing and communicating this research to a broader community. The fellow was interviewed by TU Delta about this project and wrote an article in the Kavli Newsletter describing aspects of the project for a general audience.

The project is also described in the lab website: sites.google.com/site/abbondanzierilab/home

4. Project Management

This grant has allowed the fellow to build a dynamic research group. The group encompasses two PhD students, two technicians, one master student and one bachelor student, as well as the fellow. The fellow independently manages this group and he makes all hiring, promotion, and advisory decisions within the group.

In addition, the fellow is employed as a tenure track assistant professor in the Department of Bionanoscience at TU Delft. This project has been of assistance in establishing the fellow as performing research at the highest level.

Unfortunately, because of the tight funding situation in the Netherlands and Europe, the fellow will not be able to continue his research beyond 2016. During this year he will graduate his remaining students and close his lab down.